Biotin synthase, the enzyme that catalyzes the last step of the biosynthesis of biotin, contains only [2Fe-2S]2+ clusters when isolated under aerobic conditions. Previous results showed that reconstitution with an excess of FeCl3 and Na2S under reducing and anaerobic conditions leads to either [4Fe4S]2+, [4Fe-4S]+, or a mixture of [4Fe-4S]2+ and [2Fe-2S]2+ clusters. To determine whether any of these possibilities or other different cluster configuration could correspond to the physiological in vivo state, we have used 57Fe Mössbauer spectroscopy to investigate the clusters of biotin synthase in whole cells. The results show that, in aerobically grown cells, biotin synthase contains a mixture of [4Fe-4S]2+ and [2Fe-2S]2+ clusters. A mixed 14Fe-4S]2+:[2Fe-2S]2+ cluster form has already been observed under certain in vitro conditions, and it has been proposed that both clusters might each play a significant role in the mechanism of biotin synthase. Their presence in vivo is now another argument in favor of this mixed cluster form.